Abstract
Elevated immunoglobulin (Ig) secretion by malignant cells is a characteristic feature of several malignancies including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Thus, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for these diseases. Here, we defined a novel pathway involving the oncogenic GLI transcription factors modulating IgM secretion by WM malignant cells. Initial screening of molecules involved in Ig secretion identified components of the Hedgehog-GLI pathway as modulators of this cellular process. Inhibition of smoothened (SMO), the signaling component of the Hedgehog receptor, with the pharmacological inhibitor cyclopamine had no effect on cell proliferation by XTT assay. Cyclopamine inhibition had no effect on cell viability by Annexin V/Propidium Iodide staining, but had a modest in IgM secretion by ELISA in BCWM.1 but not in MWCL-1 and RPCI-WM1 cells. Interestingly, pharmacological inhibition of GLI proteins (GLI1, 2 and 3), downstream effectors of the pathway, using GANT61 reduced the expression of GLI1 and GLI2 and had no effect on cell proliferation or viability. However, it resulted in a significant reduction in IgM secretion (40-60%) in BCWM.1 (p=0.0030), MWCL (p=0.0206) and RPCI-WM1 (p=0.0303) cells. Validation of these results using RNAi showed that out of the three GLI transcription factors, only GLI2 knockdown impaired IgM secretion by BCWM.1 (p=0.0415) and MWCL-1 (p=0.0075) cells. To further characterize the mechanism of regulation of IgM secretion by GLI2, we screened for pathways that are known to modulate Ig secretion. We identified the IL-6 receptor (IL-6R) alpha subunit as a potential mechanism mediating GLI2-IgM axis. Treatment of B-cells with GANT61 resulted in reduced IL-6R expression. Furthermore, overexpression of GLI2 in B-cells increased IL-6R expression. Conversely, GLI2 knockdown reduced IL-6R expression suggesting the involvement of IL-6R in GLI2 mediated regulation of IgM secretion. Taken together, these results identify a novel role for GLI proteins, particularly GLI2, as modulators of IgM secretion by malignant B-cells. Therefore therapies targeting this signaling axis may provide efficacy in patients with Ig-mediated diseases.
Disclosures:
No relevant conflicts of interest to declare.
